Platelet membranes are known to contain at least 3 glycoprotein components with molecular weights between 100,000-140,000 as estimated on SDS-acrylamide gels. These molecules can be labeled in intact platelets by non-permeable reagents, indicating their surface orientation. Preliminary studies in this laboratory have resulted in purification of glycoprotein from crude platelet extracts by affinity chromatography using lectin-Sepharose conjugates. SDS-acrylamide gels of the purified glycoprotein show a major PAS-positive band of molecular weight approximately 140,000 and several minor (and variable) bands. Tritiation of sialic acid residues of the glycoprotein in intact cells has confirmed its outward orientation; phosphorylation of the glycoprotein by treatment of intact cells with P32 labeled inorganic phosphate has suggested that it spans the entire membrane. The goals of the proposed project are as follows: 1) To continue purification of the platelet membrane glycoprotein (and separation of the several species) by a combination of LIS/phenol extraction, affinity chromatography and additional chromatography in detergent. 2) To study further the arrangement of the glycoprotein in the membrane by a combination of radiolabeled probes which indicate 'outside' or 'inside' orientation, and to use crosslinking reagents to study neighboring membrane molecules. 3) To study the effect of inducers and inhibitors of platelet aggregation on the arrangement and labeling reactions of the glycoprotein. 4) To study glycoprotein antigenicity in relation to immune platelet disorders. Preparation of heterologous antibody to purified glycoprotein will provide a radioimmunoassay to be used as an analytic tool in the above studies.